Diastolic dysfunction is a leading cause of morbidity and mortality, although the mechanism(s) responsible for this devastating problem remain unresolved. We determined that hRFRP-1, human RFamide-related peptide-1, slows cardiac relaxation, a component of diastole. This is an exciting finding as it identifies a potential endogenous signaling agent that could contribute to diastolic dysfunction, and therefore serve as a potential therapeutic target. Knowledge about the influence of RFamide peptides on cardiac function initially came from the invertebrate heart. The human peptide exists;however, no published studies report its function in mammalian myocytes or myocardium. A long term goal is to translate this knowledge to humans and understand the role of this peptide in relaxation. Insight gained may then be used for the future development of therapeutic strategies to lessen the impact of diastolic dysfunction. Our initial goal is to characterize the influence of hRFRP-1 on relaxation in mammalian myocytes. Longer term goals are to understand the role of hRFRP-1 on cardiac performance under physiological and pathophysiological conditions, and relevance to human cardiovascular health. This is submitted as an R21 to explore the potential for high impact research as we translate our work in Drosophila melanogaster to mammalian systems, and ultimately humans. The hypothesis tested here is hRFRP-1 slows relaxation in myocytes via (1) its C terminus and (2) binding to rfr-2. We demonstrated D. melanogaster myosuppressin (DMS) slows heart rate. DMS and hRFRP-1 C termini are identical;the C terminus is required for DMS activity. DMS acts through a GPCR, and hRFRP-1 binds to rfr-2. Our aims are to (#1) elucidate hRFRP-1 structure-activity relationship (SAR), and (#2) delineate hRFRP-1 binding in myocytes. SAR data are used to design agonists and antagonists and confirm their influence on diastolic function, and to delineate mechanisms involved in the effect of hRFRP-1 on relaxation. Ligand- receptor binding identifies the molecule that hRFRP-1 interacts with to transduce its signal. The approach used in aim 1 will be to determine the influence of substituted and truncated peptide analogs of hRFRP-1 on relaxation. The approach used in aim #2 will be to investigate rfr-2 expression in cardiac myocytes and bind a detectable hRFRP-1 analog to myocyte proteins. Our proposed research fills an important scientific gap in cardiovascular physiology;how a conserved neuropeptide acts to slow cardiac relaxation. A long-term goal is to understand the role of hRFRP-1 in human diastole and apply this knowledge for diagnosis and/or to develop future therapeutic strategies to address diastolic dysfunction in cardiovascular disease. PUBLIC HEALTH RELEVANCE The proper filling of the heart with blood is crucial to health. Problems in filling with blood may lead to heart failure causing illness and death. Our goal is to understand how blood filling is regulated by the brain and the heart to develop therapies or drugs to combat this problem.